This invention relates to an electrophoretic method of separating alkaline phosphatase isoenzymes.
The term "alkaline phosphatase" (abbreviated ALP), EC 3.1.3.1, is applied to a group of non-specific enzymes that hydrolyze monophosphoric esters of a variety of natural and synthetic compounds over a pH range of approximately 8.5 to 10.5. The different chemical species subsumed under the term alkaline phosphatase are referred to herein as isoenzymes of alkaline phosphatase. Alkaline phosphatase enzymes are found in many human tissues and in blood serum, and the isoenzyme composition of a given tissue is believed to be fixed genetically.
Determination of ALP has long been a routine clinical procedure as an aid in diagnosing various disease states. It was early recognized that identification of isoenzymes of ALP in serum can be a useful diagnostic tool for differentiating disease states characterized by increased serum ALP levels. Heat inactivation was first used to differentiate osteogenic (bone) ALP from the isoenzyme originating in hepatic (liver) tissue. In recent years, more delicate techniques for separating ALP isoenzymes have revealed several isoenzymes, some of which are present in elevated quantities in human sera even in non-pathologic conditions. For example, a "placental" ALP isoenzyme is elevated in serum during the last trimester of pregnancy; the same isoenzyme appears to be elevated in the serum of patients suffering from certain carcinomas. Likewise, the bone isoenzyme is elevated in growing children, as well as in patients suffering from certain bone diseases. Determination of ALP isoenzymes is now, therefore, important not only as a diagnostic tool, but also as a tool for basic biological and medical research.
In recent years, refinements in electrophoretic techniques have led to development of useful procedures for separating a number of ALP isoenzymes. In these procedures, a serum or tissue sample is applied to an appropriate support medium (such as agarose, polyacrylamide, cellulose acetate or starch gels) containing an appropriate electrolyte. When the gel is subjected to a differential electrical field, proteins within the sample acquire either a positive or negative charge and move toward the cathode or anode portion of the gel. Proteins in the sample are thus separated into more or less discrete fractions or bands. By the use of one or more reagents which react with the alkaline phosphatase bands to produce detectable products (such as colored products or products which have characteristic absorption bands in the ultraviolet spectrum), the bands of ALP isoenzymes are developed and identified.
Unfortunately, presently known electrophoresis techniques are generally not sensitive enough to separate and detect all of the ALP isoenzymes which may be present in a sample. The bands tend to be diffuse, lacking in intensity, and difficult to identify with particular isoenzymes. Furthermore, the procedures tend to be difficult and time-consuming to perform.